DESCRIPTION: The last five years have witnessed a hallmark achievement in the field of HIV-AIDS therapy- at least three patients have been cured of HIV after bone marrow transplant. Unfortunately, transplantation procedures tend to be very risky and restricted to those patients who are at a substantial risk for death due to associated malignancies. Direct in vivo genetic engineering of hematopoietic cells for HIV resistance would mean a big step forward in the field of HIV-AIDS gene therapy. The objective of this proposal is to develop an effective method for providing a functional cure for HIV infected individuals. The approach is based on the observations that (i) subjects lacking or heterozygous for the expression of CCR5, the viral coreceptor, can be highly resistant to HIV infection (ii) hematopoietic CD34+ stem and progenitor cells (HPCs) with a mutated version of the CCR5 gene when transplanted into a HIV patient (The Berlin patient) afforded a cure from HIV. We have developed novel peptide nucleic acids (PNA) that can form a triple helical structure specifically within the CCR5 gene. The triplex formation induces natural cellular repair-recombination pathways enabling introduction of a stop codon in the CCR5 gene when a donor DNA is supplied alongside. These pathways are error-free and can thus be used to disrupt the CCR5 gene with extremely low off-target rates and thereby expression of wild-type CCR5 protein. We propose to use biocompatible nanoparticles made from the FDA-approved polymer PLGA for encapsulating PNA and donor DNA molecules for in vivo delivery and genome editing at the CCR5 locus in hematopoietic cells. The studies in this application are directed at enabling targeted and specific delivery of newer generation PNAs and nanoparticles to human hematopoietic cells by enabling penetrance into the bone marrow following simple intravenous injection. The efficacy of the approach in prophylaxis as well as therapy will be evaluated in a new generation humanized mouse model for HIV infection. The overall goal is to establish feasibility of a new minimally invasive and innovative therapeutic paradigm for HIV-1 infection: application of triplex and nanoparticle technology for the site-directed modification of the CCR5 gene in hematopoietic cells in vivo by facile IV infusion.